Neisseria meningitidis (meningococcus) is a Gram negative bacterium frequently isolated from the human upper respiratory tract. It is a cause of serious invasive bacterial diseases (on an endemic and epidemic scale) such as bacteremia and meningitis. The incidence of meningococcal disease shows geographical, seasonal and annual differences (Schwartz, B., Moore, P. S., Broome, C. V.; Clin. Microbiol. Rev. 2 (Supplement), S18-S24, 1989). The bacterium primarily affects children from 6 months to 2 years, but also affects teenagers. The bacterium is commonly classified according to the serogroup of its capsular polysaccharide.
Most disease in temperate countries is due to strains of serogroup B and varies in incidence from 1-10/100,000/year total population—sometimes reaching higher values (Kaczmarski, E. B. (1997), Commun. Dis. Rep. Rev. 7: R55-9, 1995; Scholten, R. J. P. M., Bijlmer, H. A., Poolman, J. T. et al. Clin. Infect. Dis. 16: 237-246, 1993; Cruz, C., Pavez, G., Aguilar, E., et al. Epidemiol. Infect. 105: 119-126, 1990).
Epidemics dominated by serogroup A meningococci, mostly in central Africa, sometimes reach incidence levels of up to 1000/100,000/year (Schwartz, B., Moore, P. S., Broome, C. V. Clin. Microbiol. Rev. 2 (Supplement), S18-S24, 1989). Nearly all cases as a whole of meningococcal disease are caused by serogroup A, B, C, W-135 and Y meningococci, and a tetravalent A, C, W-135, Y capsular polysaccharide vaccine is available (Armand, J., Arminjon, F., Mynard, M. C., Lafaix, C., J. Biol. Stand. 10: 335-339, 1982). The available polysaccharide vaccines are currently being improved by way of chemically conjugating them to carrier proteins (Lieberman, J. M., Chiu, S. S., Wong, V. K., et al. JAMA 275: 1499-1503, 1996). The serogroup B capsular polysaccharide has been found to be nonimmunogenic—most likely because it shares structural similarity with host components (Wyle, F. A., Artenstein, M. S., Brandt, M. L. et al. J. Infect. Dis. 126: 514-522, 1972; Finne, J. M., Leinonen, M., Mäkelä, P. M. Lancet ii.: 355-357, 1983).
The frequency of Neisseria meningitidis infections has risen in the past few decades in many European countries. This has been attributed to increased transmission due to an increase in social activities under crowded conditions (for instance discos, swimming pools, theatres, etc.). It is no longer uncommon to isolate Neisseria meningitidis strains that are less sensitive or resistant to some of the standard antibiotics. This phenomenon has created an unmet medical need and demand for new anti-microbial agents, vaccines, drug screening methods, and diagnostic tests for this organism.
The development of effective vaccines requires reliable tests for establishing whether an effective immune response has been elicited in vaccinated individuals. For N. meningitidis serogroup A, B and C vaccines Serum Bactericidal Activity (SBA) assays are seen as being the gold standard in the field (with a four-fold increase in SBA being accepted as surrogates for protection for these serogroups) [Borrow et al., 2001, Infect Immun 69:1568-1573; Vermont et al., 2002, FEMS Immun and Med Microbiol 34:89-96]. The assay can give information on: a) whether any particular serum sample has a level (titre) of bactericidal antibodies sufficient to reach a protective threshold, b) a % seropositivity (% SP) of subjects surpassing this level, c) a % seroconversion (% SC) of subjects either i) with a ratio of SBA titres (post- to pre-immunisation sera titres) which increase beyond a particular multiple (4-fold), or ii) as for % SP if the level of SBA titres pre-immunisation in the subjects (i.e. the very young) is below the protective SBA threshold. This assay is therefore used as a primary read-out (of protection and vaccine efficacy). In addition to SBA assays IgG ELISA assays are often used as a secondary readout. These ELISA assays measure the concentration (in μg/ml) of IgG antibodies in a serum sample which bind to a meningococcal capsular polysaccharide (A, C, W135 or Y).
In A, C, W-135 (or W), and Y SBA assays the effect of respectively specific anti-serogroup A, C, W and Y Neisseria meningitidis antibodies are evaluated in the presence of complement (typically from baby rabbit [a rSBA assay], or alternatively from human sera [a hSBA assay]). A mixture of bacteria and complement is added to the sera. The meningococcal specific antibodies bind to the target cell-surface via meningococcal-specific protein or carbohydrate moieties. The C1q subunit of C1 binds to the Fc portion of the surface-bound Ig. The binding of C1q to Ig activates the classical pathway of the complement which ultimately results in death of the target cell. The bactericidal titre for each serum is expressed as the reciprocal serum dilution corresponding to 50% killing.
SBA assay methods are well-known in the art. In 1976 WHO Expert Committee on Biological Standardization recommended an SBA assay to be used to satisfy the requirements for production and release of meningococcal polysaccharide vaccines (WHO Tech. Rep. Ser. 1976, 594:72-73). Since this time Centers for Disease Control and Prevention (CDC), Atlanta, Ga., USA has researched into the various variables in an SBA assay (No. of CFU per well, assay buffer, growth of the target strain, assay incubation time, source of complement, complement concentration, and starting dilution of serum) and has recommended standard SBA assay protocols well-known and used in the field (Maslanka et al. Clin. Diag. Laboratory Immun. 1997, 4:156-167). Typically the CDC also recommends and provides meningococcal strains to be used in SBA assays in the meningococcal vaccine field. For instance for N. meningitidis serogroup C (MenC) SBA assay (WHO guidelines) a consensus has been reached with regard to the recommended assay strain to use.